Effect of dihydrotestosterone on mouse embryonic stem cells exposed to Mi Na Lee 1,† , Sang Hun Lee 2,† , Min Young Lee 2 , Yun Hee Kim 2 , Jae Hong Park 2 , Jung Min Ryu 2 , Seung Pil
Trang 1J O U R N A L O F Veterinary Science
J Vet Sci (2008), 9(3), 247256
*Corresponding author
Tel: +82-62-530-2831; Fax: +82-62-530-2809
E-mail: hjhan@chonnam.ac.kr
†
The first and second authors contributed equally to this work.
Effect of dihydrotestosterone on mouse embryonic stem cells exposed to
Mi Na Lee 1,† , Sang Hun Lee 2,† , Min Young Lee 2 , Yun Hee Kim 2 , Jae Hong Park 2 , Jung Min Ryu 2 , Seung Pil Yun 2 ,
Yu Jin Lee 2 , Mi Ok Kim 2 , Kwangsung Park 1 , Ho Jae Han 2, *
1 Department of Urology, Chonnam National University Medical School, Gwangju 501-746, Korea
2 Biotherapy Human Resources Center (BK 21), College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea
Oxidative stresses induced by reactive oxygen species (ROS)
have been shown to be involved in several physiological and
pathophysiological processes, such as cell proliferation and
differentiation Steroid hormones can protect cells against
apoptosis or induce cell proliferation by several mechanisms
Among androgenic hormones, dihydrotestosterone (DHT) is
generated by a 5 α- reduction of testosterone Unlike testosterone,
DHT cannot be aromatized to estradiol, therefore DHT is
considered a pure androgenic steroid This study was conducted
to examine the effect of DHT (10 -7 M) on H 2 O 2 (10 -3 M)
-induced injuries in mouse embryonic stem (ES) cells H 2 O 2
induced ROS generation and increased lipid peroxide
formation and DNA fragmentation These effects of H 2 O 2
were inhibited by pretreatment with DHT H 2 O 2 also increased
the phosphorylation of p38 MAPK, SAPK/JNK and nuclear
factor kappa B (NF- κB), but DHT blocked these effects
Moreover, H 2 O 2 decreased DNA synthesis and the levels of cell
cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent
kinase (CDK) 2, and CDK 4] These effects of H 2 O 2 were
inhibited by pretreatment with DHT In conclusion, DHT may
partially prevent H 2 O 2 -induced cell injury through inhibition
of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK
and NF- κB in mouse ES cells.
Keywords: DHT, dihydrotestosterone, H2O2, mouse ES cell,
oxidative stress
Introduction
Although oxygen is required for aerobic life, it may be
toxic under certain conditions [1,25,37] Oxidative stress
has been shown to play an important role in the pathogenesis
of embryo development [4,28,39] Reactive oxygen species (ROS) are forms of oxygen that result from incomplete reduction of molecular oxygen that may induce different types of cell injury, in particular, lipid peroxidation and membrane damage Although low levels of ROS play an important role in physiological functions, several studies have reported that high concentrations of ROS are cytotoxic and have been implicated in the pathological conditions such as carcinogenesis of various types of malignancies [3,52,55] The major oxygen species responsible for these oxidative stresses are hydrogen peroxide (H2O2), the free radical superoxide anion (O2-) and the hydroxyl radical (OH- ) [6] Among the ROS, H2O2 has been implicated as a cellular toxin [19,61] Since H2O2 has remarkable membrane permeability [23], intracellular H2O2 can induce detrimental effects in cells [20,41,42,49] In addition, oxidative stress on tissues and cells has been widely recognized as a key factor effecting the development of embryos [13,31,32] Therefore, the inhibition of such free radical-mediated pathology has become a central focus of research efforts targeted at the prevention or amelioration of embryo or embryonic stem (ES) cell injury However, the effects of dihydrotestosterone (DHT) on ES cells, in the context of oxidative stress, have not been thoroughly evaluated to date
DHT is a biologically active metabolite of the hormone testosterone; it is formed primarily in the prostate gland, testes, hair follicles, and adrenal glands by the enzyme
5α-reductase by reduction of the delta-4,5 double-bond of testosterone [15,24,52] DHT has been shown to be a powerful antioxidant, effectively preventing the formation
of lipid peroxides (LPO) [8] The chemical structure of DHT allows for the donation of an H+ atom to a peroxyl radical [45,60], which results in free radical scavenging and may exert additional effects by early interference or during the propagation phase of LPO It is postulated that this antioxidant activity is one mechanism by which DHT confers cardio-, hepato- and, neuro- protection Despite
Trang 2their antioxidant activities, it is not known whether DHT
prevents or alleviates the ES cell toxicity induced by
oxidative stress
ES cell lines are pluripotent cells derived from the
blastocyst-stage of mammalian embryos [12,16] These
unique cells are characterized by their capacity for prolonged
undifferentiated proliferation in culture with the potential
to differentiate into derivatives of all three germ layers
[46,47,51] In addition, these cells closely resemble their in
vivo counterparts, providing a stable in vitro model of
embryo growth and development that acts as a model for
the study of specific cell signaling systems [30,33]
Therefore, the study of mouse ES cells has provided a
versatile biological system that has led to major advances
in cell and developmental biology Many studies have used
H2O2 to investigate the mechanisms of cell injury resulting
from acute oxidative stress in various cells and tissues
[18,59] Therefore, the purpose of this study was to determine
the effect of DHT on mouse ES cells under conditions of
H2O2-induced oxidative stress
Materials and Methods
Materials
The mouse ES cells were obtained from the American
Type Culture Collection (ES-E14TG2a) The fetal bovine
serum (FBS) was purchased from Gibco (USA) DHT was
acquired from TCI (Tokyo Kasei Kogyo, Japan) The 5-(and
-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
acetyl ester (DCF-DA) was purchased from Molecular Probes
(USA) Hydrogen peroxide solutions were obtained from
Sigma-Aldrich (USA) Phospho-p38 mitogen activated protein
kinase (MAPK), p38 MAPK, phospho-c-Jun N-terminal
kinase/stress activated protein kinase (SAPK/JNK), SAPK/
JNK, and phospho-nuclear factor (NF)-κB, and NF-κB
antibodies were purchased from New England Biolabs
(UK) Cyclin D1, cyclin E, cyclin dependent protein kinase
(CDK) 2, and CDK 4 antibodies were purchased from
Santa Cruz Biotechnology (USA) Goat anti-rabbit IgG
were obtained from Jackson Immunoresearch (USA)
Liquiscint was obtained from National Diagnostics (USA)
ES cell culture
Mouse ES cells were cultured for 5 days in Dulbecco’s Modified
Eagle Media (DMEM; Gibco-BRL, USA) supplemented
with 3.7 g/l sodium bicarbonate, 1% penicillin and streptomycin,
1.7 mM L-glutamine, 0.1 mM β-mercaptoethanol, 5 ng/ml
mouse leukemia inhibitory factor and 15% FBS The cells
were grown on either gelatinized 12-well plates or a 60 mm
culture dish in an incubator maintained at 37oC in an
atmosphere containing 5% CO2 and air
Assay of intracellular reactive oxygen species
The cells were pretreated with or without DHT for 30 min and
then were treated with H2O2 for 0 to 120 min DCF-DA was used for the detection of H2O2-induced ROS, which acts as
an H2O2-sensitive fluorophore Next, 10 μM DCF-DA was added to the cells, which were then incubated in the dark for
30 min at room temperature The cells were then viewed using
a laser confocal microscope (×400; fluoview 300; Olympus, Japan), fluorescence was excited at 488 nm and emitted light was observed at 515-540 nm
To quantify the intracellular H2O2 levels, the cells treated with DCF-DA were rinsed twice with ice-cold PBS then scraped
A 100 μl cell suspension was loaded into a 96-well plate and examined using a luminometer (Victor3; PerkinElmer, Finland) and a fluorescent plate reader with excitation and emission wavelengths of 485 nm and 535 nm, respectively
DNA fragmentation assay
The cells were pretreated with or without DHT for 30 min and then were treated with H2O2 for 24 h The cells were then suspended in lysis buffer (10 mmol/l Tris-HCl pH 7.5,
10 mmol/l EDTA pH 8.0, 0.5% Triton-X) The cell lysates were treated with 200 μg/ml proteinase K at 60oC for 6 h followed by DNA extraction using phenol-chloroform The extracted DNA was precipitated using isopropyl alcohol, then digested with 10 μg/ml TE-RNase at 37oC for
1 h After digestion, the product was electrophoresed on a 1% agarose gel stained with ethidium bromide and photographed under ultraviolet light
Measurement of lipid peroxides
The cells were pretreated with or without DHT for 30 min and then were treated with H2O2 for 0 to 120 min The level of LPO
in the monolayer cells was determined by measuring the malondealdehyde content according to the description of
Ohkawa et al [48] The mouse ES cells were washed twice with
PBS, then harvested and sonicated One hundred microliters
of sonicated cells were mixed with 8% SDS (100 μl), 0.8% 2-thiobarbituric acid (TBA; 200 μl) and 20% acetic acid (200 μl) The mixture was heated to 95°C for 60 min, then cooled in ice water To extract nonspecific red pigment, a n-butanol-pyridine mixture (15 : 1 vol/vol, 1 ml) was added, then shaken vigorously and centrifuged at 4,000 rpm for 10 min The upper organic layer was measured using spectrofluorometry
at an emission wavelength of 553 nm with an excitation wavelength of 515 nm The 1,1,3,3-tetraethoxypropane was used as a standard, and the values of LPO for the samples were expressed as nmol/mg protein Taurine was added to cell mixtures to prevent any initiation of membrane lipid peroxidation during the assay An addition of taurine to the standard 1,1,3,3-tetraethoxypropane or a control sample did not affect the color development with the TBA (data not shown)
Trypan blue exclusion assay
The cells were pretreated with or without DHT for 30 min and
Trang 3Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H 2 O 2 -induced oxidative stress 249
Fig 1 Effect of hydrogen peroxide (H2O2) on cell injury A, B: The cells were incubated in H2O2 treated for 0-60 min and then Dichlorofluorescein-sensitive cellular reactive oxygen species (ROS) was observed by confocal microscopy and luminometer C: Cells were treated with H2O2 for 0 to 120 min and then lipid peroxide formation was measured The values are reported as the means ± SE of three
independent experiments with triplicate dishes *p ＜ 0.05 vs control D: Cells were treated with H2O2 for 0 to 24 h and then the effects
of HO-induced DNA fragmentation were investigated The example shown is a representative of three independent experiments
then were treated with H2O2 for 24 h and then washed twice with
PBS The cells were then detached from the culture dishes
using a 0.05% trypsin and 0.5 mM EDTA solution, and this
action was quenched with a soybean trypsin inhibitor (0.05
mg/ml) Subsequently, a 0.4% (w/v) trypan blue solution
(500 μl) was added to the cell suspension and the cells were
counted using a hemocytometer while keeping a separate
count of the blue cells The cells that failed to exclude the dye
were considered non-viable; therefore, the data is expressed
as a percentage of viable cells Cell injury was assessed by
measuring the LDH activity in the medium using a LDH
assay kit (Iatron Lab, Japan) The level of LDH released is
expressed as a percentage of the control value
[ 3 H] thymidine incorporation
The [3H] thymidine incorporation experiments were carried
out as described by Brett et al [11] In this study, the cells were
cultured in one well until they reached 50% confluence, at which
time they were washed twice with PBS, then maintained in
serum-free DMEM with all supplements and incubated for
24 h The cells were pretreated with or without DHT for 30
min and then were treated with H2O2 for 24 h Next, 1 μCi of
[methyl-3H] thymidine (specific activity: 74 GBq/mmol, 2.0 Ci/mmol; Amersham Biosciences, UK) was added to the cultures and the incubation continued for 1 h at 37oC The cells were then washed twice with PBS, fixed in 10% trichloroacetic acid (TCA) at 23oC for 15 min, then washed twice with 5% TCA The acid-insoluble material was dissolved in 0.2 N NaOH for 12 h at 23oC Aliquots were removed and their radioactivity determined using a liquid scintillation counter (LS 6500; Beckman, USA) All values are means ± SE of triplicate experiments Values were converted from absolute counts to a percentage of the control to allow for comparison between experiments
Western blot analysis
The cell homogenates (20 μg protein) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred
to nitrocellulose membranes After the blots were washed with TBST [10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20], the membranes were blocked with 5% skim milk for 1 h and incubated with the appropriate primary antibodies
at the dilutions recommended by the supplier The membrane was then washed and the primary antibodies detected using
Trang 4Fig 2 Effect of dihydrotestosterone (DHT) on hydrogen peroxide (H2O2)-induced apoptotic cell death A: Dichlorofluorescein-sensitive cellular ROS was measured by confocal microscopy The cells were treated with DHT for 30 min prior to H2O2 treatment and were incubated for 60 min, and then the cellular levels of H2O2 were measured B: Lipid peroxide formation was measured after the cells were pretreated with DHT for 30 min prior to H2O2 treatment and were incubated for 120 min C, D: The cells were pretreated with DHT (10-7 M) for
30 min prior to H2O2 treatment for 24 h, then LDH release and cell viability were measured The values are reported as the mean ± SE
of three independent experiments with triplicate dishes *p ＜ 0.05 vs control; **p ＜ 0.05 vs H2O2 alone E: Cells were pretreated with DHT for 30 min prior to a 24 h H2O2 treatment and DNA fragmentation was assessed as described in the Materials and Methods section The example shown is a representative of three independent experiments
goat anti-rabbit IgG or goat anti-mouse IgG conjugated to
horseradish peroxidase The bands were visualized using enhanced
chemiluminescence (Amersham Pharmacia Biotech, UK)
Statistical analysis
The results are expressed as the mean ± SE All experiments
were analyzed by ANOVA, which was followed in some
cases by a comparison of the treatment and the control
samples using the Bonnferroni-Dunn test A p value ＜
0.05 was considered significant
Results
Effect of DHT on H 2 O 2 -induced cell injury
We determined the H2O2-induced ROS generation in mouse
embryonic stem cells using DCF-DA (10-5M), a fluorescent probe of intracellular H2O2 Production of ROS was detected in the cells exposed to H2O2 10-3 M for a variety of times (0-60 min) by the DCF fluorescence intensity in the ES cells As shown Figs 1A and B the level of ROS generation was increased after ≥ 10 min In addition, H2O2 treatment significantly increased LPO formation after ≥ 30 min compared with controls as shown in Fig 1C In order to examine the effect of H2O2 on cell injury, the ES cells were incubated for a variety of times (0-24 h) with H2O2 exposure and the level of DNA fragmentation was examined The
H2O2 increased the level of DNA fragmentation after ≥
16 h (Fig 1D) However, the H2O2-induced cell injury was prevented by DHT (10-7 M) pretreatment As shown in Figs 2A and B increase in the ROS generation and LPO
Trang 5Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H 2 O 2 -induced oxidative stress 251
Fig 3 Effect of hydrogen peroxide (H2O2)-induced p38 mitogen activated protein kinase (MAPK) and c-Jun N-terminal kinase/stress activated protein kinase (SAPK/JNK) phosphorylation A, B: The cells were pretreated with dihydrotestosterone (DHT) for 30 min prior to the H2O2 treatment for 60 min and phosphorylation of p38 MAPK and SAPK/JNK was measured The phosphorylated p38 MAPK and SAPK/JNK were then detected using Western blot analysis The lower panels of A and B depict the bars showing the mean ± SE of three experiments for each condition determined by densitometry relative to β-actin *p ＜ 0.05 vs Control, **p ＜ 0.05 vs H2O2 alone
formation by H2O2 were inhibited by DHT In addition, as
shown in Figs 2C and D the increase of LDH release and
the decrease of cell viability, with H2O2, were inhibited by
DHT This is further supported by the assessment of the
DNA fragmentation experiments (Fig 2E)
Effect of DHT on H 2 O 2 -induced activation of p38
In order to assess the involvement of MAPKs in
H2O2-induced cell injury, the cells were treated with H2O2
for 30 min and then phosphorylation of MAPKs was measured
by Western blot analysis As shown in Figs 3A and B, H2O2
increased p38 MAPK and SAPK/JNK phosphorylation,
which was inhibited by DHT In the next step, the involvement
of NF-κB activation in H2O2-induced cell injury was
evaluated As shown in Fig 4A, H2O2-induced NF-κB
activation was inhibited by DHT treatment In addition,
NF-κB phosphorylation was attenuated by pretreatment
with SB 203580 (a p38 MAPK inhibitor) and SP 600125 (a
SAPK/JNK inhibitor, 10-6 M) (Fig 4B)
Effect of DHT on H 2 O 2 -induced DNA synthesis
regulation
To investigate the effect of DHT on H2O2-induced
increase of cell cycle regulatory protein expression, the
mouse ES cells were pretreated with DHT for 30 min prior
to adding the H2O2 The H2O2-induced decrease of cell
cycle regulatory protein (cyclin D1, cyclin E, CDK 2, and
CDK 4) expression levels were recovered by pretreatment with DHT in the mouse ES cells (Figs 5A, B, C and D) In addition, as shown in Fig 5E, the maximum decrease in the level of [3H] thymidine incorporation was observed following 24 h of H2O2 exposure (30% decrease vs
Control; *p ＜ 0.05) However, this inhibitory effect of
H2O2 on DNA synthesis were blocked by pretreatment with DHT, SB 203580 (p38 MAPK inhibitor, 10-6 M), SP
600125 (SAPK/JNK inhibitor, 10-6 M), SN 50 (NF-κB phosphorylation inhibitor, 10-6 M), and Vitamin C (antioxidant, 10-3 M)
Discussion
In the present study, we showed that DHT prevented
H2O2-induced cell injury likely by the downregulation of p38 MAPK, SAPK/JNK and NF-κB in mouse ES cells Oxidative stress induced by ROS is a well known cause of several physiological and pathophysiological processes including cell proliferation and differentiation [26,50] ROS, which include the superoxide anion (O2- ), H2O2, OH, and singlet oxygen (1O2) are formed by incomplete reduction of molecular oxygen ROS may cause different types of cell injury, particularly lipid peroxidation and membrane damage [6] H2O2 is often used as a model compound to induce oxidative stress in cell systems [54] The antioxidant properties
of DHT have not been fully studied and multiple mechanisms may be involved in its action [10,21,22] LPO formation in
Trang 6Fig 4 Effect of dihydrotestosterone (DHT) on hydrogen peroxide (H2O2)-induced nuclear factor kappa B (NF-κB) phosphorylation A: The cells were pretreated with DHT for 30 min prior to the H2O2 treatment for 60 min and then the phosphorylation of NF-κB was measured B: The cells were pretreated with SB 203580 (p38 mitogen activated protein kinase inhibitor, 10-6 M) or SP 600125 (c-Jun N-terminal kinase/stress activated protein kinase (SAPK/JNK) inhibitor, 10-6 M) before H2O2 treatment; the phosphorylated NF-κB was then detected using Western blot analysis The lower panels of A and B depict the bars showing the mean ± SE of three experiments for each condition determined from densitometry relative to β-actin *p ＜ 0.05 vs Control, **p ＜ 0.05 vs H2O2 alone
biological membranes is a free radical-mediated event and
is regulated by the availability of substrates in the form of
polyunsaturated fatty acids, pro-oxidants which promote
peroxidation, and inhibited by antioxidants such as β-carotene
and superoxide dismutase [14,27,55] Elevated levels of
LPO have been linked to cell injury such as the increase in
permeability to ions and eventually the disruption of the cell
membrane leading to release of cell organelles [14,27,55]
The results of this study showed that exposure to 1 mM H2O2
generated LPO, and DHT attenuated the LPO formation
The findings of this study demonstrated that the protective
effects of DHT were associated with the inhibition of LPO
generation, which is consistent with the results of previous
reports [40,62] Bennett et al [7] reported that testosterone
with a keto group at the C3 position, showed a mild
preventive effect compared to DHT Thus we suggest that
the protective effects of DHT are due to its basic chemical
properties as a hydrophobic phenolic molecule
H2O2-induced oxidative stress mediates the phosphorylation
of protein kinases through a series of signal cascades, such
as activation of MAPKs and, a nuclear factor-κB [44] The
MAPKs proteins are mediators of signal transduction from
the cell surface to the nucleus and play a major role in
triggering and coordinating gene responses [36] In
addition, phosphorylation of JNK and p38 MAPK has been
shown to play a role in cellular differentiation and
inflammatory responses [17,29,35] Antioxidants have been shown to attenuate the activation of MAPKs signaling [29,58], thereby indicating that the MAPK signaling pathway is an important target of ROS The levels of intracellular ROS were increased as the result of the addition of extracellular H2O2, which subsequently increased p38 MAPK and SAPK/JNK phosphorylation In the present study, DHT treatment was observed to inhibit the effects of H2O2 on MAPKs phosphorylation, which supports the role of DHT in this process In addition, H2O2 activated NF-κB is one of the key regulatory molecules in oxidative stress-induced cell injury and the activation of NF-κB has an important regulatory role in cell proliferation and oncogenesis [2,43,56] NF-κB is commonly activated
by oxidants, including H2O2 [34] In this study, we demonstrated that NF-κB was activated by H2O2 treatment
In addition, activated NF-κB was blocked by SB 203580 (p38 MAPK inhibitor) and SP 600125 (SAPK/JNK inhibitor) These results suggest that NF-κB is a down stream target of JNK and p38 MAPK In addition, DHT treatment inhibited the H2O2-induced activation of these signaling molecules Moreover, the p38 protein of the MAPK family has been shown to modulate NF-κB activation [9,57] The inhibition
of H2O2-induced phosporylation of MAPKs proteins may result in the prevention of downstream events such as activation of NF-κB [44] Therefore, we suggest that the
Trang 7Effect of dihydrotestosterone on mouse embryonic stem cells exposed to H 2 O 2 -induced oxidative stress 253
Fig 5 Effect of dihydrotestosterone (DHT) on hydrogen peroxide (H2O2)-induced decrease of DNA synthesis A-D: The cells were pretreated with DHT for 30 min prior to H2O2 treatment for 24 h and then Western blot analysis for cyclin D1, cyclin E, cyclin dependent protein kinase (CDK) 2, and CDK 4 was carried out The lower panels of A, B, C, and D depict the bars showing the mean ± SE of three experiments for each condition determined by densitometry relative to β-actin *p ＜ 0.05 vs Control, **p ＜ 0.05 vs H2O2 alone E: The cells were pretreated with DHT, SB 203580, SP 600125, SN 50, and vitamin C for 30 min prior to H2O2 treatment for 24 h and then [3H] thymidine incorporation was conducted as described in the Materials and Methods section The values are reported as the mean
± SE of three independent experiments with triplicate dishes *p ＜ 0.05 vs control; **p ＜ 0.05 vs H2O2 alone
significant inhibition of H2O2-induced phosphorylation of
MAPKs by DHT might be responsible for the inhibitory
effects on the activation of the transcription factor NF-κB
Cyclin-dependent kinases (CDK 2 and CDK 4) are
enzymes responsible for the coordinated progression of the
cell cycle [53] CDKs are activated by their association
with the D-type cyclins (cyclin D1, D2 and D3); cyclin D1
plays an important role in the G1 phase of cell cycle
progression in association with CDK 4 [53] H2O2 is
known to induce cell cycle arrest and apoptosis in various
cell types cultured in vitro [5] MAPKs also lead to the
increase of cell cycle regulatory proteins [cyclin D1, cyclin
E, CDK 2, and CDK 4] [38] In this study, H2O2 decreased
the expression of cell cycle regulatory proteins However,
DHT inhibited the effect of H2O2-induced decrease of cell cycle regulatory protein expression Moreover, H2O2 decreased DNA synthesis However, DHT inhibited the effect of the H2O2-induced decrease of DNA synthesis Moreover, the H2O2 decrease of DNA synthesis was not only inhibited by DHT treatment but also other antioxidants (vitamin C), SB 203580, SP 600125, and SN
50 These findings suggest that H2O2-induced activation of p38 MAPK, SAPK/JNK and NF-κB are involved in the mechanism associated with decreased DNA synthesis (Fig 6)
In conclusion, the results of this study confirmed the protective effects of DHT were associated with the inhibition of p38 MAPK, SAPK/JNK and NF-κB signaling pathways by free
Trang 8Fig 6 The hypothesized model for the signal pathways involved in the protective effects of DHT for the H2O2-induced mouse embryonic stem cell injury DHT, dihydrotestosterone; ROS, reactive oxygen species; MAPK, mitogen activated protein kinase; SAPK/ JNK, c-Jun N-terminal kinase/stress activated protein kinase; NF-κB, nuclear factor kappa B; IκB, inhibitory kappa B; CDK, cyclin dependent protein kinase
radical scavenging These data demonstrate that DHT appears
to have powerful cytoprotective activity against H2O2-induced
ES cell injury
Acknowledgments
This work was supported by Stem Cell Research Program
(M10641450001-06N4145-00110), Ministry of Education,
Science and Technology, and Korea Research Foundation
Grant funded by the Korean Government (MOEHRD)
(KRF-2004-042-E00095)
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